The present invention features a method of inhibiting vital reproduction and a preparation useful for accomplishing this inhibition. More particularly, the present invention features a method of in vivo or in vitro inhibition of viral reproduction by interfering at the nucleic acid level. The method used is non-toxic to the other cells of the body and therefore has advantages relative to most chemotherapeutic viral agents.
Viruses are infectious agents having a central core of nucleic acid surrounded by an outer core of protein or lipoprotein. Viruses require a host cell as an integral part of their reproductive process. One subclass of viruses are the enveloped viruses, which act by fusion of the protein with cell membranes rather than injection of the whole virus into the cell. These enveloped viruses can have either RNA or DNA as the nucleic acid, although RNA viruses are more common. Examples of these enveloped viruses include orthomyxoviruses such as influenza virus, coronaviruses, herpesviruses, the paramyxoviruses such as Sendai and Newcastle disease viruses, retroviruses and HIV. These enveloped viruses normally have a glycoprotein/lipid exterior with "receptors" that mediate virus attachment to target cells and nucleic acid penetration into these cells. These virus envelopes are strong enough to provide effective protection to transport nucleocapsids.
One difference between the enveloped viruses, as compared with certain other viruses, is their mode of infectious activity. The enveloped viruses, which may include some retro-viruses such as HIV-1, inject the nucleic acid into the host cell by fusion of the virus lipoproteins or proteins with the cell membrane rather than penetration of the virus protein through the membrane wall. This fusion of the virus with the cell membrane is normally mediated by a fusion protein which has a structure with a high rate of preservation from species to species. Fundamental Virology, B. N. Fields and D. M. Knipe, editors, Raven Press, N.Y., Chapter 4, 18-23, 27-28, 33-36.
The development of phospholipid and glycolipid based lipid vesicles, generally called liposomes, was primarily as models for cell membrane structure. There has recently been an explosion in the number of papers reporting the use of these liposomes to test viral fusion. Most of this work has been carried out using either Sendai virus or Influenza A virus. For example, experiments with Influenza virus show that dextran sulphate inhibits the fusion of the virus with liposomes. See Luscher-Mattli and Gluck, Anti-Viral Research 14, pp. 39-50 (1990). In addition, much work has also been carried out on the effect of charge and rate of fusion between Sendal virus and phospholipid vesicles. See, e.g., Nir, Klappe, and Hoekstra, Biochemistry 25, pp. 6261-6266 (1989); and Stegmann, Nir and Wilschut, Biochemistry 28, pp. 1698-1704 (1989). Similarly, Huang et al., U.S. Pat. No. 4,789,633, used pH sensitive phospholipid materials to show the effect of pH on vesicle fusion.
The articles reporting vesicle/virus fusion with these model systems describe experiments carried out with phospholipid and/or glycolipid materials since they are used to model the action of cellular membranes. Normally, unilamellar vesicles are utilized as their cellular model. Since the experiments are directed to the mechanism of the vital insertion of nucleic acid, the reproductive activity of the fusion product is not normally tested. As such, there appears to be little data to determine whether these fusion products (or hybrids) could still infect other cells.
One problem in dealing with viruses is that they are not susceptible to classic antibiotics. This makes treatment of patients, as well as clinical experiments, difficult. Although certain chemotherapeutic agents and other chemoprophylactic drugs such as Acyclovir and Amantadine have been used against various viruses, they are not universally effective. Similarly, although it has been theorized that interferon might be useful in treating viral diseases, this has also not been overly successful.
Much of the clinical virus work has been directed to vaccines. However, little work has been carried out on vaccines using vesicles, and even less on anti-vital vaccines containing vesicles. To the extent there have been tests, they have been carried out using "killed" viruses. As such, this anti-viral work has used the vesicles solely as adjuvants. One example of this anti-viral vaccine is the Newcastle disease vaccine sold by Immunogenetics, Inc., a sister company of the assignee of the present application. This vaccine uses killed Newcastle disease virus in conjunction with non-phospholipid paucilamellar lipid vesicles.
As an adjunct to the work in connection with development of this Newcastle disease vaccine, the present inventors tested the effect of using live rather than killed viruses in conjunction with the vesicles as an immunizing agent. The purpose behind this type of work is that in certain circumstances, the live viruses make better immunizing agents than killed viruses. Surprisingly, not only did the live viruses work as immunizing agents, it appears that fusion took place between the non-phospholipid paucilamellar vesicles and the virus protein envelope. Although fusion between phospholipid vesicles and virus coatings were known, it is unexpected that non-phospholipid vesicles could fuse since there is a large difference between the properties of a phospholipid membrane such as is used in a conventional liposome and the non-phospholipid membranes. Phospholipids have a dual carbon chain structure as compared with the singular carbon chains used in the non-phospholipid vesicles.
Even more interesting, the fusion product of the paucilamellar non-phospholipid vesicles and the virus appeared not to be infectious; that is, the fusion appeared to denature the nucleic acid of the virus. Accordingly, this has opened a new realm of speculation and experimentation for the inventors.
The results uncovered by the present inventors in connection with the Newcastle disease vaccine has led to the possibility of a new method for prevention and treatment for vital infections. Accordingly, an object of the invention is to provide a treatment for vital infection using paucilamellar lipid vesicles.
Another object of the invention is to provide a method of preparing an anti-viral immunizing agent by combining an enveloped virus with paucilamellar lipid vesicles.
A further object of the invention is to provide a method of treating viral infections without affecting other cells.
Still further object of the invention is to provide a method of making a vaccine useful in treating viral infections.
These and other objects and features of the invention will be apparent from the following description and the claims.